In vitro and in vivo gene expression studies of cartilage-like tissue engineered construct using a combination of transiently transfected human osteoarthritic chondrocytes and tissue engineering technique

Autor: Shaban Munirah, Shukri Norhamiza, Zulkifly Ahmad
Rok vydání: 2016
Předmět:
Zdroj: Frontiers in Bioengineering and Biotechnology. 4
ISSN: 2296-4185
1383-1453
DOI: 10.3389/conf.fbioe.2016.02.00010
Popis: Purpose: To evaluate the formation of cartilage-like tissue engineered constructs (TECs) using human osteoarthritic chondrocytes overexpressed with SOX9 gene seeded on poly(lactic-co-glycolic acid) (PLGA) with and without fibrin scaffolds through cartilaginous genes expression analysis. Methods: Upon approval by the IREC18; NMRR-12-1383-14531 and IIUM/IACUCApproval/2015/[5][22], six cartilage samples were obtained from consented patients after joint replacement surgery. The cells were isolated, cultured and transfected with pcDNA3-SOX9 using Lipofectamine 2000™. Prefabricated disc-shaped porous PLGA with and without fibrin were used as scaffolds. The ‘cells-scaffolds’ TECs were formed and cultured for 3-week and implanted subcutaneously at the dorsum of athymic mice for 4-week. Collagens I, II, IX, X, XI, SOX9 and aggrecan expression were evaluated using a qualitative two-step reverse-transcriptase PCR. GAPDH and β-actin genes were used as internal controls. Results: Presence of cartilaginous markers can be detected in all TECs with various expression intensity. Collagen II, the cartilage-specific marker was down-regulated in vitro but re-expressed in vivo. Collagen I, X, SOX9 and aggrecan were steadily expressed in all TECs. Although collagen IX and XI are closely associated with collagen II, their expressions were almost untraceable except for few cases. Presence of GAPDH and β-actin genes indicated the reliability of the analysis. Conclusion: Cartilage-like TECs have been successfully formed based on genes expression analysis.
Databáze: OpenAIRE
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